The invention relates to oligoribonucleotide analogs with terminal 3xe2x80x2xe2x80x943xe2x80x2 and/or 5xe2x80x2xe2x80x945xe2x80x2 internucleotide linkages. This modification stabilizes the molecules altered in this way, including ribozymes, without adversely altering their properties, including, where appropriate, catalytic activities.
Nucleic acid fragments whose sequence is complementary to the coding or sense sequence of a messenger RNA or to the codogenic strand of the DNA are called antisense oligonucleotides. Oligonucleotides of this type are increasingly being used for inhibiting gene expression, usually from the viewpoint of medical therapy, in vitro, in cell culture systems and in vivo (1. E. Uhlmann, A. Peyman, Chem. Rev. 90 (1990) 543-584; 2. J. Goodchild, Bioconjugate Chem. 1 (1990) 165-187; 3. L. Whitesell, A. Rosolen, L. Neckers, Antisense Research and Development 1 (1991) 343).
Variations of the antisense principle are:
I. Triple helix-forming oligonucleotides: nucleic acid fragments which are able to bind to the DNA double strand to form a triple helix and which modulate gene expression by inhibiting transcription (J. Chubb and M. Hogan, TIBTECE 10 (1992) 132-136).
II. Ribozymes: Ribonucleic acid fragments with enzymatic activity which comprises cleavage of the target RNA, for example an zRNA, after the specific binding of the ribozyme by the same (T. R. Cech, J. Am. Med. Assoc. 260 (1988) 3030).
For it to be possible to employ antisense oligonucleotides, triple helix-forming oligonucleotides and ribozymes in biological systems it is, however, necessary for the following conditions to be fulfilled (E. Uhlmann, A. Peyman, Chem. Rev. 90 (1990) 543-584):
1. on the one hand they must be readily soluble in water, but on the other hand easily pass through the lipophilic cell membrane,
2. they must be sufficiently stable to degradation inside the cell, i.e. stable to nucleases,
3. they must form stable hybrids with in intracellular nucleic acids at physiological temperatures,
4. the hybridization must be selective; the difference in the dissociation temperature to an oligonucleotide which results in a mispairing must be sufficiently large for it still to be possible for the latter to be specifically washed out,
5. in the case of ribozymes, the catalytic activity must be retained.
Unmodified oligonucleotides and, in particular, unmodified oligoribonucleotides are subject to extensive nucleolytic degradation. This is why at an early stage investigations were carried out into the structural modification of oligonucleotides so that they better meet the abovementioned requirements, in particular are better protected against nuclease degradation. For this purpose a large number of oligonucleotide analogs has been prepared, in some cases with enormous synthetic effort (1. E. Uhlmann, A. Peyman, Chem. Rev. 90 (1990) 543-584; 2. J. Goodchild, Bioconjugate Chem. 1 (1990) 165-187).
It was recently shown that 3xe2x80x2-3xe2x80x2- and/or 5xe2x80x2-51-terminally linked oligodeoxynucleotides and their analogs have distinctly increased stability against nucleolytic degradation (1. B. Seeliger, A. Frxc3x6hlich, M. Montenarh: Nucleosides+Nucleotides 10 (1991) 469-477; Z. H. Rxc3x6sch, A. Frxc3x6hlich, J. Ramalho-Ortigao, J. Flavio, M. Montenarh, H. Seeliger: EP 0464638A2). Surprisingly, it has now been found that the same type of terminal linkage, which is easily accessible synthetically
a) is also able to stabilize the very much more labile oligoribonucleotides to nucleases,
b) is able to stabilize ribozymes (oligoribonucleotides with particular sequence requirements) to nucleases without impairing the catalytic activity,
c) is additionally able to stabilize oligoribonucleotides and ribozymes which have been protected from nucleases by chemical modification.
The invention therefore relates to oligoribonucleotides of the formula I 
in which
R1 is hydrogen or a radical of the formula II 
R2 is hydrogen or a radical of the formula III 
xe2x80x83but where at least one of the radicals R2 or R2 is a radical of the formula II or III;
B is a base such as, for example, natural bases such as adenine, thymine, cytosine, guanine or unnatural bases such as, for example, purine, 2,6-diaminopurine, 7-deazaadenine, 7-deazaguanine, N4,N4-ethanocytosine or their prodrug forms;
R3 is, independently of one another, OH, hydrogen, O(C1-C18)alkyl, O(C2-C18)alkenyl, F, NH2 or its prodrug forms and N3, but at least one R3 radical is different from H, and R1 is preferably OH, hydrogen, O(C1-C6) alkyl, O(C2-C6)alkenyl, F, NH23.
W and Wxe2x80x2 are, independently of one another, oxygen or sulfur;
Z and Zxe2x80x2 are, independently of one another, Oxe2x88x92; Sxe2x88x92; C1-C18,-alkoxy, preferably C12-C8-alkoxy, particularly preferably C1-C3-alkoxy, especially methoxy; C1-C18-alkyl, preferably C1-C8-alkyl, particularly preferably C1-C3-alkyl, especially methyl; NER4 with R4=preferably C1-C18-alkyl, particularly preferably C1-C8-alkyl, especially C1-C4-alkyl or C1-C4-alkoxy-C1-C6-alkyl, preferably methoxyethyl; NR4R5, in which R4 is as defined above and R5 is preferably C1-C18,-alkyl, particularly preferably C1-C8-alkyl, especially C1-C4-alkyl, or in which R4 and R5 are, together with the nitrogen atom carrying them, a 5-6-membered heterocyclic ring which may additionally contain another hetero atom from the series comprising O, S and N, such as, for example, morpholine;
where X is OH, H, F, Cl, Br, NH2, N3, Oxe2x80x94C(O)xe2x80x94(C1-C18)-alkyl, Oxe2x80x94C(O)xe2x80x94(C2-C18)-alkenyl, Oxe2x80x94C(O)xe2x80x94(C2-C18)alkynyl, Oxe2x80x94C(O)xe2x80x94(C6-C18)aryl, Oxe2x80x94(C1-C18)-alkyl, Oxe2x80x94(C2-C18)-alkenyl, Oxe2x80x94(C2C18)alkynyl, Oxe2x80x94(C6-C18)aryl, P(O)YYxe2x80x2, where Y and Yxe2x80x2 are defined as Z and Zxe2x80x2. R3 and X in formula II can togetherxe2x80x2form a cyclic phosphoric diester.
X is preferably OR, H, F, particularly preferably OH, and
n is an integer from 5-60, preferably 10-40 and especially preferably 15-25,
and their physiologically tolerated salts.
Aryl is to be understood to mean in this connection, for example, phenyl, phenyl substituted (1-3 times) by C1-C6-alkyl, C1-C6-alkoxy and/or halogen.
The oligoribonucleotides of the formula I are preferred. Furthermore preferred are oligoribonucleotides of the formula I in which R2 is a radical of the formula III and R1 is hydrogen; R1 or R2 is a radical of the formulae II and III respectively; or R2 is hydrogen and R1 is a radical of the formula II, where either W or Z in the latter case is not oxygen.
Furthermore, particular mention may be made of oligoribonucleotides of the formula I in which W is oxygen, or Z and W are both oxygen.
Furthermore, particular mention may be made of oligoribonucleotides of the formula I whose base sequence B1, B2, . . . Bn corresponds to the sequence requirements for ribozymes.
Emphasis should be placed in this connection on hammerhead ribozymes (for example Uhlenbeck, Nature 328 (1987) 596; Haseloff, Gerlach, Nature 334 (1988) 585), the hairpin ribozymes (for example Hampel et al., Nucl. Acids. Res. 18 (1990) 299), the human hepatitis xcex1-virus ribozyme (for example Branch, Robertson, Proc. Natl. Acad. Sci. USA 88 (1991) 10163) and the external guide sequence for RNase P (for example Forster, Altman, Science 249 (1990) 783), but very especially the hammerhead ribozymes.
Very particularly preferred oligoribonucleotides of the formula I are those in which R2 is a radical of the formula III and R1 is hydrogen.
Furthermore, mention may be made of oligoribonucleotides of the formula I which are additionally substituted by groups which favor intracellular uptake, which act in vitro or in vivo as reporter groups, and/or groups which, on hybridization of the oligoribonucleotide onto biological DNA or RNA, interact with these DNA or RNA molecules with binding or cleavage.
Examples of groups which favor intracellular uptake are lipophilic radicals such as alkyl radicals, for example with up to 18 carbon atoms, or cholesteryl, or thiocholesteryl (E. Uhlmann, A. Peyman, Chem. Rev. 90 (1990) 543-584; J. Goodchild, Bioconjugate Chem. 1 (1990) 165-187; B. Oberhauser, E. Wagner, Nucl. Acids Res. 20 (1992) 533; C. MacKellar et al., Nucl. Acids. Res. 20 (1992) 3411) or conjugates which utilize natural carrier systems such as, for example, bile acids or peptides for the appropriate receptor (for example receptor-mediated endocytosis).
Examples of reporter groups are fluorescent groups (for example acridinyl, dansyl, fluoresceinyl) or chemiluminescent groups such as, for example, acridinium ester groups.
Examples of oligonucleotide conjugates which bind to and/or cleave nucleic acids are to be found in the following references. (E. Uhlmann, A. Peyman, Chem. Rev. 90 (1990) 543-584; J. Goodchild, Bioconjugate Chem. 1 (1990) 165-187; Helene, Toulme, Biochim. Biophys. Acta 1049 (1990) 99).
Conjugate partners are, inter alia, acridine, psoralen, chloroethylaminoaryl, phenanthridine, azidophenacyl, azidoproflavine, phenazine, phenanthroline/Cu, porphyrin/Fe, benzo[e] pyridoindole, EDTA/Fe (Mergny et al., Science 256 (1992) 1681).
The characteristic structural modification of the oligoribonucleotides according to the invention comprises the internucleotide linkages at both ends of the chain being altered, i.e. being 3xe2x80x2xe2x80x943xe2x80x2 or 5xe2x80x2xe2x80x945xe2x80x2 linkages in place of biological 3xe2x80x2-5xe2x80x2 linkages. It has been found, surprisingly, that this minimal structural modification suffices to stabilize such compounds against nuclease degradation without adversely altering other properties, for example enzymatic activities.
As described hereinafter, the only slight structural modification results in a hybridization behavior which is almost identical to that of the biological oligoribonucleotides. This also results in the general applicability of these compounds as inhibitors of gene expression.
The compounds of the formula I are prepared in the same way as the synthesis of biological oligonucleotides in solution or, preferably, on a solid phase, where appropriate with the assistance of an automatic synthesizer. The invention therefore additionally relates to a process for the preparation of the oligoribonucleotides of the formula I, which comprises
a) reacting a nucleotide unit with 3xe2x80x2- or 5xe2x80x2-terminal phosphorus(III) or phosphorus(V) groups or its activated derivative with another nucleotide unit with a 3xe2x80x2- or 5xe2x80x2-terminal free hydroxyl group, or
b) assembling the oligonucleotide by fragments in the same way, eliminating where appropriate one or more protective groups temporarily introduced to protect other functionalities in the oligonucleotides obtained according to a) or b), and converting the oligonucleotides of the formula I obtained in this way where appropriate into their physiologically tolerated salt.
The starting component employed for the solid-phase synthesis for preparing oligoribonucleotides with terminally inverted 3xe2x80x2-3xe2x80x2 linkage is a support resin to which the first nucleoside monomer is attached via the 5xe2x80x2-OH group. This component is prepared using a support resin prepared by methods known from the literature (T. Atkinson, M. Smith in Oligonucleotide Synthesis, M. J. Gait (ed), 35-49 (1984)), preferably silica gel or controlled pore glass which is functionalized with amino groups. It is reacted with a nucleoside derivative which is protected on the nucleoside base and on the 3xe2x80x2-OH group and which has previously been converted into the 5xe2x80x2-p-nitrophenylsuccinate. The base-protective groups preferably employed are acyl groups, for example benzoyl, isobutyryl or phenoxyacetyl. The 3xe2x80x2 position is preferably protected by the dimethoxytrityl protective group, which can be introduced as described by M. D. Matteucci, M. H. Caruthers, Tetrahedron Letters 21 (1980), pages 3243-3246.
Further assembly of the oligoribonucleotide chain up to the penultimate chain member takes place by methods known from the literature (Beaucage, Iyer, Tetrahedron 48 (1992) 2223), preferably using nucleoside 3xe2x80x2-phosphorous ester amides or nucleoside 3xe2x80x2-H-phosphonates protected on the 5xe2x80x2-OH group by dimethoxytrityl groups. The 2xe2x80x2-hydroxyl group is preferably protected by the tert-butyldimethylsilyl group (M. Lyttle et al., J. Org. Chem. 56 (1991) 4608; Scaringe et al., Nucl. Acids Res. 18 (1990) 5433). The 2xe2x80x2-amino group (synthesis of compounds with R3=NH2) is preferably protected using the trifluoroacetyl group (Benseler et al., Nucleosides and Nucleotides 11 (1992) 1333). The last chain member employed is again a nucleoside 5xe2x80x2-phosphorous ester amide or a nucleoside H-phosphonate protected on the 3xe2x80x2-OH group, preferably using dimethoxytrityl. The preparation of an oligoribonucleotide chain of this type with terminally inverted internucleotide linkages is depicted diagrammatically hereinafter. (Phosphoramidite cycle for the preparation of oligonucleotides with 3xe2x80x2xe2x80x943xe2x80x2 and 5xe2x80x2xe2x80x945xe2x80x2 linkages at the ends.) Oligoribonucleotides with 3xe2x80x2xe2x80x943xe2x80x2 or 5xe2x80x2xe2x80x945xe2x80x2 linkages are prepared correspondingly. 
Methods known from the literature are likewise used to incorporate 2xe2x80x2-modified ribonucleotide units such as, for example, 2xe2x80x2-O-alkyl 2xe2x80x2-deoxyribonucleotides (Iribarren et al., Proc. Natl. Acad. Sci. USA 87 (1990) 7747; Sproat, Lamond in Oligonucleotides and Analogues: F. Eckstein, Ed., IRL Press, Oxford 1991); 2xe2x80x2-F- and 2xe2x80x2-NH2-2xe2x80x2-deoxyribonucleotides (Benseler et al., Nucleosides and Nucleotides 11 (1992) 1333; Pieken et al., Science 253 (1991) 314; Olsen et al., Biochemistry 30 (1991) 9735).
The oligoribonucleotides undergo terminal labeling for structure and sequence analyses, as described in Example 4 hereinafter. This takes place by radioactive labeling, preferably with the aid of 5xe2x80x2-xcex332P-ATP/polynucleotide kinase. This radioactive labeling takes place on the free 5xe2x80x2-OH group, i.e. at the opposite end of the nucleotide chain from an oligonucleotide with only biological 3xe2x80x2-5xe2x80x2 linkages.
The sequences with 3xe2x80x2-3xe2x80x2 inversion have a 5xe2x80x2-OH group at both ends and are therefore in some cases phosphorylated at both ends.
The oligonucleotides of the formula I are used for chemical hybridization methods which are based on addition onto double- or single-stranded nucleic acids or their cleavage for the regulation or suppression of the biological function of nucleic acids, and for the selective suppression of the expression of viral genome functions and for the prophylaxis and therapy of viral infections, for the suppression of oncogene function and for the therapy of cancers.
The behavior of an oligoribonucleotide of the formula I which has been assembled according to the invention and dissolved in blood serum can be regarded as a measure of the stability in vivo. The general test is described in Example 4. The oligoribonucleotides according to the invention are degraded much more slowly than the 3xe2x80x2-5xe2x80x2 oligoribonucleotides.
Example 5 demonstrates that the oligoribonucleotides according to the invention which meet the sequence requirements for hammerhead ribozymes do not differ in their enzymatic activity from the unmodified ribozymes.